Plant materials and reagents
Commercially grown PCK was obtained from Gangwon-do agricultural research and extension services in Korea (April, 2014). The plants were identified by Emeritus Professor H.J. Chi at Seoul National University, and voucher specimens were deposited in the Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University in with voucher number RIC-2014-NP-0415. Fresh PCK was dried at 45 °C in a drying oven and then stored at room temperature. A PTP1β (human, recombinant) drug discovery kit was purchased from BIOMOL® International LP (Plymouth meeting, PA). Sodium chloride, p-nitrophenyl phosphate (pNPP), and dithiothreitol were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA) for use as synthetic substrates. All other chemicals and reagents used were of analytical grade.
Extraction, fractionation and isolation
Dried PCK (1.0 kg) was ground and extracted with 35 % ethanol for 8 h at room temperature. The total filtrate was concentrated to dryness in vacuo at 40 °C. The PCK 35 % ethanol extract powder (500 g) was applied to an open glass column packed with Diaion HP-20 and eluted with water to wash any sugars or impure components. The packing was then suspended in water and partitioned sequentially with n-Hexane, CH2Cl2, EtOAc, and n-BuOH, leaving a residual aqueous fraction. The EtOAc fraction showed inhibitory activity on PTP1β, hence 5.5 g of extract was subjected to C18 gel column chromatography eluted with water and increasing methanol in an H2O-MeOH gradient system (95:5 → 0:50, v/v) to obtain 7 compounds. Also, the n-BuOH fraction (1.0 g) was subjected to high speed counter current chromatography (HSCCC). The HSCCC system employed in the present study was a Model TBE-1000A HSCCC (Shanghai Tauto Bio technique, Shanghai, China) with 3 multilayer coil columns connected in series and was equipped with a 50-mL sample loop. The inner diameter of the PTFE tubing was 1.8 mm, and the total volume capacity was 1000 mL. The b-value of the preparative column varied from 0.42 at the internal layer to 0.63 at the external layer. The rotation speed of the apparatus was regulated using a speed controller in the range of 0-600 rpm. The HSCCC system was equipped with a Model Hitachi L-6200 intelligent pump (Hitachi, Tokyo, Japan), Model TOPAZ dual UV monitor operating at 520 nm, and Model ECOMAC-ECOM Acquisition and Control (version 0.97). The upper phase, consisting of a mixture of n-BuOH:acetic acid:water (4:1:5, v/v/v) was used as the stationary phase, while the lower phase was used as the mobile phase. The mobile phase was pumped at 2.5 mL/min, while centrifugation was carried out at 400 rpm. As a result, 12 compounds were isolated and identified by 1H & 13C NMR spectra (COSY, HMBC, HMQC and DEPT) and LC-MS/MS.
Assay method of PTP1β inhibitory activity
A PTP1β (human, recombinant) drug discovery kit was purchased from BIOMOL® International LP (Plymouth meeting, PA). Enzymatic activity was measured using pNPP, as described previously. To each of the 96-wells in a microtiter plate (final volume: 100 μL) was added 2 mM pNPP and PTP1β (0.05-0.1 ng/well) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M sodium chloride, 1 mM EDTA, and 1 mM dithiothreitol, with or without test compounds. Following incubation at 37 °C for 30 min, the reaction was terminated with 10 M sodium hydroxide. The amount of p-nitrophenol produced was estimated by measuring the absorbance at 405 nm. The non-enzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1β enzyme.
Kinetics of PTP1β by active compounds
Inhibition kinetics studies were carried out in the absence and presence of active compounds with various concentrations of pNPP (0.1, 0.5 and 1.0 mM) as substrate. The initial rate was determined on the basis of the rate of increase in absorbance at 405 nm. The Michaelis-Menten constant (K
m
) and maximal velocity (Vmax) of PTP1β were determined by Lineweaver-Burk Plot analysis for competitive inhibition, and the intercept on the vertical axis for noncompetitive inhibition [16].
Extraction process
The dried PCK (1.0 g) was accurately weighed and placed in a capped tube and mixed with 10 mL of 35 % ethanol. After wetting the plant material, the tube containing the suspension was immersed at 37 °C in a water bath and irradiated for the predetermined for 30 min. After extraction, the sample was centrifuged at 3000 rpm for 3 min. The supernatant was collected and diluted with eluent. All samples were filtered through 0.45 μm syringe filter.
Experimental design for RSM
The effects of the four independent processing parameters (extraction temperature (X1, °C), extraction time (X2, hour), solid-liquid ratio (X3, %), and solvent volume (X4, 1:X)), on the dependent variables were investigated using RSM. The CCD for RSM required only five levels, coded as -2, -1, 0, +1, +2. The total number of experiments designed was 27 based on the five levels and a four-factor experimental design, with five replicates at the central conditions of the design for estimation of a pure error sum of squares. The dependent variables were TPC (Y1), TAC (Y2), and EY (Y3). The model equation for the response (Y) to the three independent variables (X1, X2, X3 and X4) is given in the following equation:
$$ \boldsymbol{Y}={\boldsymbol{\beta}}_0+{\displaystyle \sum_{\boldsymbol{i}=1}^2}{\boldsymbol{\beta}}_{\boldsymbol{i}}{\boldsymbol{X}}_{\boldsymbol{i}}+{\displaystyle \sum_{\boldsymbol{i}=1}^2}{\boldsymbol{\beta}}_{\boldsymbol{i}\boldsymbol{i}}{\boldsymbol{X}}_{\boldsymbol{i}}^2+{\displaystyle \sum_{\boldsymbol{i}}}\ {\displaystyle \sum_{\boldsymbol{j}=\boldsymbol{i}+1}}{\boldsymbol{\beta}}_{\boldsymbol{i}\boldsymbol{j}}{\boldsymbol{X}}_{\boldsymbol{i}}{\mathbf{X}}_{\mathbf{j}} $$
Total polyphenol content determination
Total polyphenol content (TPC) was determined according to the Folin Denis Method with a slight modification. The extract was double-diluted and 100 μL of the diluted sample was mixed with 50 μL Folin Ciocalteu’ reagent and 300 μL of 2 % (w/v) sodium carbonate. After incubating the samples at room temperature for 1 h, 1 mL water was added before measuring the absorbance at 750 nm. The calibration curve was obtained using gallic acid in the same manner as done for the sample (R2 = 0.999). Results were expressed as mg of gallic acid equivalent (GAE) per g of dried weight.
Total anthocyanin content determination
Total anthocyanin (TAC) was used to indicate the contents of anthocyanin extracted from PCK. TAC was determined using a pH differential method. Absorbencies were read at 530 and 700 nm. Pigment content was calculated as cyanidin-3-glucoside (C3G) using an extinction coefficient (ε) of 26,900 and a molecular weight of 449.2 and expressed as mg cyanidin-3-glucoside equivalent (C3GE) per 100 g of dried weight [17].
Determination of extraction yield
The PCK extracts were concentrated in an efficient centrifugal concentration system (EZ-2 plus, Genevac and UK) and the difference in weight corresponds to the soluble solid (total extract yield) of the dried PCK.
Data analysis
All calculations and analyses were performed using statistical analysis system (SAS, SAS Institude Inc., NC, USA, version 9.1) software and Sigma plot (Systat Software Inc., USA, version 11). Inhibition rates were calculated as percentages (%) with respect to the control value and IC50 value was estimated from the least-squares regression line of the logarithmic concentration plotted against inhibitory activity.